Project Summary We will use the `incubation of craving' model to study a major challenge in treating cocaine addiction, that is, the persistence of vulnerability to cue-induced relapse even after long periods of abstinence. In this model, rats exhibit progressive intensification (incubation) of cue-induced craving after withdrawal from extended-access cocaine self-administration. Incubation of craving also occurs in humans. Previously, we showed that Ca2+- permeable AMPARs (CP-AMPARs; homomeric GluA1) accumulate in synapses on medium spiny neurons (MSN) of the nucleus accumbens (NAc) core after ~30 days of withdrawal. Thereafter, their activation is required for expression of incubated cocaine craving. Thus, understanding CP-AMPAR plasticity may provide clues about how the intensity of craving is regulated. Work from our lab and others has shown that cocaine withdrawal is associated with multiple adaptations that predict reduced activity or Ca2+ signaling in NAc MSN. However, little is known about how this might be linked to CP-AMPAR insertion. A promising candidate is a form of homeostatic plasticity involving retinoic acid (RA) signaling. Work in hippocampal neurons has shown that Ca2+ levels associated with ongoing synaptic transmission are sufficient to suppress RA synthesis. However, after a period of inactivity, RA synthesis is disinhibited. This increases GluA1 translation, enabling a homeostatic increase in synaptic strength via insertion of homomeric GluA1 CP-AMPARs. CP-AMPAR accumulation in the NAc core during incubation of craving may likewise involve reduced Ca2+ (see above) and we recently showed that it is associated with increased GluA1 translation. Based on these similarities to RA-mediated homeostatic plasticity in hippocampus and on other data, we hypothesize that cocaine withdrawal is associated with decreased Ca2+ signaling in MSN, leading to disinhibition of RA synthesis, increased GluA1 translation and increased synaptic levels of homomeric GluA1 CP-AMPARs. Aim 1 will test the relationship between excitatory transmission, RA synthesis, and GluA1 translation in cultured NAc MSN (co-cultured with cortical cells to restore glutamate inputs). Aim 2 will use fiber photometry to test the hypothesis that baseline Ca2+ in NAc core MSN is reduced during cocaine withdrawal, although it may transiently increase during cue-induced seeking tests and this increase may be augmented as incubation of craving occurs. Transgenic rats expressing Cre recombinase in D1 or A2a receptor-positive MSN (A2a is a marker for D2 MSN) will enable both MSN populations to be studied. In Aim 3, whole-cell patch clamp recordings in identified D1 or A2a NAc core MSN will determine if acute manipulation of RA signaling alters CP-AMPAR levels and if protein translation is involved. Then, we will determine if knocking down a critical RA synthetic enzyme in NAc core during cocaine withdrawal attenuates CP-AMPAR accumulation and maintenance of incubated craving after long withdrawal. Conversely, we will knock down the enzyme that degrades RA in saline rats to determine if a long-term increase in RA is sufficient to increase CP-AMPARs. Overall, our studies will use state-of-the-art tools to test a conceptually novel hypothesis for incubation of craving.